Claudins are tight junction proteins that determine paracellular permeability and probably form the lining of the paracellular pore. In the renal tubule, they play important roles in electrolyte and acid-base handling. The long-term goal of this proposal is to determine the structural and functional determinants of ionic selectivity in the paracellular pore. We propose to use claudin-2 as a model cation-selective pore. Our overall HYPOTHESIS is that the paracellular cation pore is a relatively wide, water-filled cylinder, the wall of which is formed from the extracellular domains. The side chains of acidic residues within the extracellular domain contribute negatively charged sites that line the pore. We hypothesize that monovalent cations permeate the pore by partially dehydrating and are then stabilized either by discrete electrostatic binding sites, or simply by the altered dielectric environment within the pore. The SPECIFIC AIMS are to: (1) Characterize the biophysical properties of the claudin-2 paracellular pore and model the permeation of cations through the pore;(2) Identify acidic residues in the extracellular domains of claudin-2 that determine paracellular cation selectivity and characterize their role;(3) Identify other pore-lining residues in claudin-2 by cysteine mutagenesis and determine their location and function METHODS to be used include stable transfection of MDCK I cells with claudin-2, Ussing chamber studies of diffusion potential and conductance, interference with negatively charged residues by pH and carboxyl modifying reagents, site-directed mutagenesis of extracellular residues, and probing of cysteine-substituted residues by methanethiosulfonate reagents. The data obtained will lead to a structural model to explain the function of claudin-based pores. This may shed light on disorders such as hypertension and renal salt-wasting and lead to the development of drugs that could regulate paracellular permeability of the renal tubule and correct electrolyte disorders.